Crystal Structure of Argonaute and Its Implications for RISC Slicer Activity

Argonaute proteins and small interfering RNAs (siRNAs) are the known signature components of the RNA interference effector complex RNA-induced silencing complex (RISC). However, the identity of “Slicer,” the enzyme that cleaves the messenger RNA (mRNA) as directed by the siRNA, has not been resolved. Here, we report the crystal structure of the Argonaute protein from Pyrococcus furiosus at 2.25 angstrom resolution. The structure reveals a crescent-shaped base made up of the amino-terminal, middle, and PIWI domains. The Piwi Argonaute Zwille (PAZ) domain is held above the base by a “stalk”-like region. The PIWI domain (named for the protein piwi) is similar to ribonuclease H, with a conserved active site aspartate-aspartate-glutamate motif, strongly implicating Argonaute as “Slicer.” The architecture of the molecule and the placement of the PAZ and PIWI domains define a groove for substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage.

[1]  J. M. Thomson,et al.  Argonaute2 Is the Catalytic Engine of Mammalian RNAi , 2004, Science.

[2]  Michael Sattler,et al.  Nucleic acid 3′-end recognition by the Argonaute2 PAZ domain , 2004, Nature Structural &Molecular Biology.

[3]  D. Patel,et al.  Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain , 2004, Nature.

[4]  Phillip D Zamore,et al.  The RNA-Induced Silencing Complex Is a Mg2+-Dependent Endonuclease , 2004, Current Biology.

[5]  Thomas Tuschl,et al.  RISC is a 5' phosphomonoester-producing RNA endonuclease. , 2004, Genes & development.

[6]  E. Sontheimer,et al.  A Dicer-2-Dependent 80S Complex Cleaves Targeted mRNAs during RNAi in Drosophila , 2004, Cell.

[7]  T. Du,et al.  RISC Assembly Defects in the Drosophila RNAi Mutant armitage , 2004, Cell.

[8]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[9]  B. Simon,et al.  Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain , 2003, Nature.

[10]  Ming-Ming Zhou,et al.  Structure and conserved RNA binding of the PAZ domain , 2003, Nature.

[11]  Ji-Joon Song,et al.  The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes , 2003, Nature Structural Biology.

[12]  Henning Urlaub,et al.  Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi , 2002, Cell.

[13]  Phillip D Zamore,et al.  Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. , 2002, Molecular cell.

[14]  T. Tuschl,et al.  Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate , 2001, The EMBO journal.

[15]  P. Zamore,et al.  ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway , 2001, Cell.

[16]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[17]  A. Bateman,et al.  Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain. , 2000, Trends in biochemical sciences.

[18]  S. Kim,et al.  Crystal structure of archaeal RNase HII: a homologue of human major RNase H. , 2000, Structure.

[19]  W. Reznikoff,et al.  Three-dimensional structure of the Tn5 synaptic complex transposition intermediate. , 2000, Science.

[20]  S. Hammond,et al.  An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells , 2000, Nature.

[21]  Merkel,et al.  Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant. , 1999, Biochemistry.

[22]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[23]  P. Rice,et al.  Structure of the bacteriophage Mu transposase core: A common structural motif for DNA transposition and retroviral integration , 1995, Cell.

[24]  T. Steitz,et al.  Crystal structure of lac repressor core tetramer and its implications for DNA looping. , 1995, Science.

[25]  T. Steitz,et al.  Recombining the structures of HIV integrase, RuvC and RNase H. , 1995, Structure.

[26]  A. Engelman,et al.  Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. , 1994, Science.

[27]  Haruki Nakamura,et al.  Atomic structure of the RuvC resolvase: A holliday junction-specific endonuclease from E. coli , 1994, Cell.

[28]  K. Morikawa,et al.  Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8 Å resolution: Proof for a single Mg2+‐binding site , 1993, Proteins.

[29]  U. Wintersberger.  Ribonucleases H of retroviral and cellular origin. , 1990, Pharmacology & therapeutics.